Application of the CAPG and PTGIS genes in scoliosis detection kit is preparedTechnical field
The present invention relates to biomedicine field, and in particular to CAPG and PTGIS genes are preparing scoliosis detection reagentApplication in box.
Background technology
Scoliosis (Scoliosis), also known as scoliosis, are that a class is bent with backbone side and with Vertebral rotationComplex three-dimensional deformity of spine.According to pathogenic factor, 3 major classes can be classified as：Congenital scoliosis, syndrome scoliosisAnd idiopathic scoliosis.Congenital scoliosis is caused by vertebral column development deformity；Syndrome scoliosis is by godCaused by muscle disease, skeletal diseases, connective tissue disease and neurofibromatosis etc.；The definite cause of disease of idiopathic scoliosisIt is unclear, correlative study think gene genetic factor, grow with hormone, connective tissue lesion, muscle systems it is abnormal,Central nervous system exception, effect of epiphysin etc. all may be relevant with AIS morbidity.Wherein, adolescent idiopathic backbone sideCurved (Adolescent Idiopathic Scoliosis, AIS) is most commonly seen, accounts for the 80% of whole scoliosis.Teenager is specialThe incidence of disease of the hair property scoliosis in -16 years old 10 years old teenagers is higher, is after eyesight abnormality, obesity, phimosis and social mentalityThe fifth-largest common disease after obstacle.Although AIS does not generally cause serious pathological state in addition to deformity is showed, severe chest is curvedThe decline of the heart, PFT may be caused, cause the deformity on obvious build, or even influence life-span.
Current treatment method is rescued primarily directed to deformity, including clinical observation, Brace Treatment and lateral bending is exceeded45 ° of persons carry out operative treatment, and treatment is complicated, has often left deformity of spine and backbone moving obstacle, effect is undesirable.Meanwhile, AISIf patient is without examination, in first go to a doctor, the curved average angle of master is 38 °, already close to the upper limit for carrying out Brace Treatment.Therefore, early screening and diagnosis to AIS can strive for the chance of more expectant treatments for patient, reduce operation probability.At presentAIS diagnosis is with examination mainly or by the physical examination to patient's outward appearance symmetry and imageological examination, and this can make a lotThe teenager of low-risk receives unnecessary radiological examination, and this examination means specificity is poor.With medical geneticsLearn and Medical Molecular Biology research deepening continuously and improves, the research of AIS Medical Molecular Biology mechanism also successively byReport.Some relative biomarkers are found that during molecular biology research is carried out to AIS, such as calcium adjusts eggIn vain, leptin, Serum osteopontin, solubility CD44, SH3GL1 etc. in peripheral blood, but effect and conclusion still need to further checking.Therefore, get on to study the AIS causes of disease from gene expression dose, continually look for AIS early screenings new, that specificity is good and diagnosisLabel is significant.
The content of the invention
In order to realize the early detection of Adolescent idiopathic scoliosis, early treatment, it is an object of the invention to provideA kind of kit for scoliosis early screening.
To achieve the above object, present invention firstly provides a kind of kit for scoliosis early screening, the examinationThe primer that agent box includes being used to detect CAPG genes includes such as SEQ ID NO:1 and SEQ ID NO:Nucleotide sequence shown in 2.
It is preferred that, the primer that the kit also includes being used to detect PTGIS genes includes such as SEQ ID NO:3 and SEQID NO:Nucleotide sequence shown in 4.
It is preferred that, the kit also includes dNTPs, Taq enzyme, Mg2+With PCR reaction buffers.
It is preferred that, the kit can detect CAPG and PTGIS genes in biological sample by real-time PCROr the expression of its expression product comes diagnosis of vertebral lateral bending, CAPG genes and PTGIS the genes table in biological sampleUp to downward.
Further, the present invention provides a kind of ELISA kit for scoliosis early screening, the kit bagInclude the specific antibody of anti-CAPG and PTGIS albumen.
It is preferred that, CAPG the and PTGIS protein antibodies are monoclonal antibody or polyclonal antibody.The CAPG andThe specific antibody of PTGIS albumen include complete antibody molecule, any fragment of antibody or modification such as chimeric antibody,ScFv, Fab, F (ab') 2, Fv etc..As long as the fragment can retain the binding ability with CAPG or PTGIS albumen.
It is preferred that, the antibody can be obtained from commercial channels, and a series of methods known in the art can also be usedPrepare.For example, people CAPG and the PTGIS albumen or its antigen fragment of purifying are injected into animal body to produce Anti-TNF-αBody.Equally, the cell of expression people CAPG and PTGIS albumen or its antigen fragment may also be used for causing animal immune and producingAntibody.Monoclonal antibody can be prepared with hybridoma technology.
It is preferred that, CAPG the and PTGIS protein antibodies are monoclonal antibody, CAPG the and PTGIS protein antibodies canFor mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, preferably mouse source antibody.Anti-human CAPG and PTGIS that the present invention is usedAlbumen mouse resource monoclonal antibody is prepared by Shanghai Fan Ke bio tech ltd.
It is preferred that, CAPG the and PTGIS protein antibodies are the antibody of horseradish peroxidase-labeled.
It is preferred that, the kit also includes the anti-coated ELISA Plate of CAPG rabbit polyclonal antibodies, anti-PTGIS rabbit polyclonalsThe coated ELISA Plate of antibody, protein standard substance CAPG and PTGIS, tmb substrate solution, dilution, lavation buffer solution, termination are moltenLiquid.Preferably, stop bath composition is 2M H2SO4。
It is preferred that, the scoliosis is Adolescent idiopathic scoliosis.
Beneficial effects of the present invention are as follows：The invention discloses a kind of the gene C APG and PTGIS related to scoliosis,And further confirm CAPG the and PTGIS genes or its expressing protein in Adolescent idiopathic scoliosis patient derived biological sampleMiddle expression is lowered.The generaI investigation of scoliosis is carried out in teenager using CAPG and PTGIS genes, solves and examines in the prior artThe problem of disconnected measure Sensitivity and Specificity difference, the infringement of x-ray radiation is decreased, can fast and effectively accomplish AIS morningPhase diagnoses, and develops for predictive diagnosis AIS, even provides treatment to treat AIS in biology level from now onTarget spot and important evidence.
Brief description of the drawings
Scoliosis patient's CAPG and PTGIS down regulation of gene expression in embodiment 2 in Fig. 1 present invention.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodimentIn the conventional meanses that are well known to those skilled in the art of used technological means.
The experimental method of unreceipted actual conditions in embodiment, usually this area conventional method, such as according to normal conditionThe condition in such as Sambrook et al., molecular cloning, laboratory manual (third edition) (Science Press, 2002), or according toCondition proposed by reagent manufacturing firm.
Technical scheme is specifically included：20 Adolescent idiopathic scoliosis clinical samples and 15 are compareedSample carries out high-flux sequence, carries out genescreen with reference to bioinformatics method, picks out candidate gene CAPG and PTGIS,Do not have CAPG, PTGIS report related to Adolescent idiopathic scoliosis in existing research, further, inventor is carried outMolecular biology method checking, it was confirmed that CAPG and PTGIS expresses downward, its phase in scoliosis patient derived biological sampleClose preparation and can be used for diagnosis Adolescent idiopathic scoliosis.
CAPG the and PTGIS genes of the present invention are knowns before making the present invention, and its essential information is as follows：
Genbank accession number：CAPG GeneID:822, PTGIS GeneID:5740 derive from human genome.
CAPG (capping actin protein, gelsolin like, actin-modulating protein) is located at No. 2 dyeingOn body, it is Gelsolin superfamily members, belongs to the actin binding protein in cytoskeletal protein together with Gelsolin.Gelsolin is had found that it passes through the phase with actin most prior to 1979 by Yin and Stossel in the pulmonary macrophage of rabbitInteraction, is played to accuracy controlling functions such as the polymerizations, depolymerization and shearing of actin, and participates in Cellular Signaling Transduction Mediated, withCell regulation and control etc. are in close relations.It is used as Ga2+Dependence actin binding protein, CAPG is adjusted by capping, cutting to microfilamentControl the length of actin.Studies have found that, in fibroblast and endothelial cell CAPG be overexpressed can cause cellMotility strengthens.
PTGIS (prostaglandin I2 synthase, prostaglandin I synthase) be Cytochrome P450 superfamily intoMember.Cytochrome p450 protein is monooxygenase, participates in drug metabolism, the synthesis of cholesterol, steroids or other lipids.SoAnd, it based on sequence similarity rather than functional similarity is considered as Cytochrome P450 superfamily member that this protein, which is,.The memebrane protein catalysis prostaglandin H2 of endoplasmic reticulum is converted into prostaglandin (prostacyclin I2), is a kind of effective vasodilatorAnd platelet aggregation inhibitor.The physiological antagonist of prostacyclin and thromboxane A2 is unbalance to cause miocardial infarction, apoplexy and arteryThe generation of atherosis.
The experimental method specifically studied mainly includes following components：
1. utilize level of the high-flux sequence method to blood sample CAPG and the PTGIS gene of 20 scoliosis patientsWith carrying out comparison in difference in 15 check samples.
2.qRT-PCR verifies the expression of scoliosis patient's CAPG and PTGIS gene
Expression of the CAPG and PTGIS genes in scoliosis patient and control group is detected using RT-PCR method, and testedDemonstrate,prove the gene and express down-regulated gene for scoliosis.
3. the detection kit assembling of scoliosis
The kit of the scoliosis of the present invention includes consisting of part：
(1) tissue sample extracts total serum IgE reagent；
(2) Reverse Transcription；
(3) quantitative PCR reagent.
4. the ELISA detection kit of scoliosis is assembled and used
(1) preparation of conventional reagent during ELISA is tested；
(2) prepared by polyclonal antibody；
(3) coating of ELISA Plate；
(4) preparation of enzyme labelled antibody；
(5) assembling of kit.
The clinical practice of 5.ELISA kits
The ELISA kit prepared using the present inventor is detected scoliosis patient to be made a definite diagnosis and detected with actual clinicalCompare that the validity of ELISA kit is determined.Specifically include CAPG and PTGIS albumen in measure subject's blood sampleExpression quantity, and be compared with CAPG and PTGIS expressing quantities in normal blood sample, it is that clinician quick and precisely graspsThe morbid state and coincident with severity degree of condition of patient, takes the control prece of more personalized to provide support in time.
The nucleotides full length sequence or its fragment of CAPG the and PTGIS genes of the present invention can generally use PCR TRAPs, againGroup method or artificial synthesized method are obtained.Once obtain relevant sequence, it is possible to had in large quantity with recombination methodClose sequence.
At present, it is already possible to encode the DNA sequence dna of the albumen (or its fragment) of the present invention by chemical synthesis completely.SoThe DNA sequence dna can be introduced into afterwards in the various DNA moleculars in this area (such as carrier) and cell.The fragment of albumen of the present invention exceptOutside being produced with recombination method, solid phase technique also can use to be produced (Stewart et al., (1969) by direct synthetic peptideSoliod-Phase Peptide Synthesis, WH Freeman Co., San Francisco；Merrifield J.(1963)J.Am Chem.Soc 85:2149-2154).Synthetic protein can be carried out by hand or automatically in vitro.For example,Peptide can be automatically synthesized with Applied Biosystems 431A types peptide synthesizer (Foster City, CA).It can distinguishEach fragment of chemical synthesis albumen of the present invention, is then chemically connected to produce the molecule of total length.
When " scoliosis " used herein is not explained, Adolescent idiopathic scoliosis is typically refered in particular to.
Terms used herein " biological sample " includes but is not limited to the samples such as blood, serum, saliva, urine, synovia, cartilageProduct.Biological sample for the present invention derives from any tissue sample (such as interverbebral disc, articular process, spinal cord, the vertebra of any subjectOther flesh or blood sample) or cell sample (such as Gegenbaur's cell, cartilage cell or blood cell samples) can be made according to the inventive methodWith.Can therefrom obtain these samples and according to the inventive method using these samples subject's example including but not limited to withoutThe subject of symptom subject, performance or more scoliosis symptom, clinical diagnosis are the subject with scoliosis, are susceptible to suffer fromScoliosis subject (if any the subject of scoliosis family history, have scoliosis genetic predisposition subject andLife style makes the subject that scoliosis possibility is suffered from its susceptible scoliosis or raising), suspect with scoliosis byExamination person, just receiving scoliosis treatment subject, with scoliosis and it is non-receive scoliosis treatment subject, openingDoctor (such as doctor) is defined as health or subject (i.e. normally) without scoliosis, the subject for having cured scoliosis, justControl the subject of its scoliosis and have not been diagnosed as the subject of scoliosis.
The high-flux sequence of embodiment 1 screens difference expression gene
1st, sample
Choose the scoliosis patient gone to a doctor during in October, 2012 in December, 2015 in BJ Union Hospital's orthopaedics, diseaseExample group collects 20 altogether, and all patients have typical clinical manifestation, are made a definite diagnosis through x-ray inspection.Control derives from same time orthopaedicsOther diseases patient in hospital, collects 15 altogether.Patient is the teenager of -18 years old 10 years old.Gather the blood of all research objectsLiquid sample, numbers rearmounted -80 DEG C of low temperature refrigerators and preserves.All clinical samples of this research, to patient know and inform simultaneouslyPass through through this Hospital Ethical Committee.
2nd, Total RNAs extraction is carried out to blood sample
UsingLS(Invitrogen:RNA extractions, experiment behaviour 10296-010) are carried out to the blood sample of collectionMake to carry out by product description, the concrete operations of every group of experiment are as follows：
The new blood being collected into is taken, 3 times of volume erythrocyte cracked liquids are added, room temperature placement 10 minutes after mixing, 10,000rpm is centrifuged 1 minute.Thoroughly inhale and abandon supernatant, collect leukocyte cell pellet.
(1) 1mLTrizol, room temperature preservation 5 minutes are entered；
(2) chlorination imitates 0.2mL, uses forced oscillation centrifuge tube, fully mixes, and room temperature is placed 3-5 minutes；
(3) 12000rpm high speed centrifugations draw upper strata aqueous phase (inhaling 70%) into another new centrifuge tube pipe after 15 minutes, noteMeaning should not be drawn onto the interface between two layers of aqueous phase.New pipe is moved into, -20 DEG C of isometric pre- cold isopropanols are added, it is fully reverse mixedIt is even, it is placed in 10 minutes on ice；
(4) supernatant carefully is discarded after 12000rpm high speeds were from 15 minutes, 75% is added in 1mL/mL Trizol ratioDEPC ethanol washes paint precipitation (4 DEG C of preservations), washes paint sediment, vibration is mixed, and 8000rpm is centrifuged 2 minutes at 4 DEG C.Discard ethanolLiquid, places 2 minutes fully to dry precipitation at room temperature, adds the treated water dissolving precipitations of DEPC；
(5) with Nanodrop2000 ultraviolet specrophotometers measurement RNA purity and concentration, freeze in -80 DEG C.RNA massCriterion：The OD260/OD280 values of RNA samples is between 1.7-2.2；Total serum IgE electrophoresis pattern has clearly 28S, 18S barBand；70 DEG C of water-baths be incubated 1 hour after electrophoresis pattern be incubated with water-bath before collection of illustrative plates no significant difference.
3rd, the quality analysis of RNA sample
Agarose gel electrophoresis after RNA is extracted, the RNA sample that can be extracted with preliminary judgement from electrophoresis result it is up-to-standard withIt is no, if to can be used for further transcriptome analysis.And then RNA sample is detected by NanoDrop1000 spectrophotometersExtraction situation, the sample requirement of RNA-seq sequencings：OD260/OD280 is 1.8-2.2.
4th, high-flux sequence
Microarray dataset is the high-flux sequence platforms of HiSeq 2500 of Illumina companies, carries out high flux transcript profile depthSequencing, we use Fast-QC (http after sequencing://www.bioinformatics.babraham.ac.uk/projects/Fastqc/) software carries out total evaluation to the quality of sequencing data, includes the quality Distribution value of base, the position point of mass valueCloth, G/C content, PCR duplication contents, kmer frequency etc..In differential genes expression analysis, according to obtainingFPKM values, using internationally recognized algorithm EBSeq carry out differential screening.Wherein, during screening, LOG2FC>1 or<- 1, FDR<0.05.In order to be better understood from the function of difference expression gene, we difference expression gene has been carried out Gene Ontology andSignal path is analyzed, and carries out functional annotation and protein interaction network analysis to difference expression gene, in view of above numberAccording to the result of analysis, with reference to document, we have screened difference expression gene CAPG and PTGIS, CAPG and PTGIS genes in backboneExpress and lower in lateral bending blood samples of patients sample.
The qRT-PCR of embodiment 2 verifies the expression of scoliosis patient's CAPG and PTGIS gene
1st, material
Choose the scoliosis patient 15 gone to a doctor during in October, 2012 in December, 2015 in BJ Union Hospital's orthopaedicsExample, all patients have typical clinical manifestation, made a definite diagnosis through x-ray inspection.Other diseases that control is in hospital from same time orthopaedicsPatient, collects 8 altogether.Patient is the teenager of -18 years old 10 years old.Gather after the blood sample of all research objects, numberingPut -80 DEG C of low temperature refrigerator preservations.All clinical samples of this research, to patient know and inform and entrusted through this hospital ethicsMember can pass through.
2nd, method
2.1 pairs of blood samples carry out Total RNAs extraction, the extracting method of be the same as Example 1.
2.2 reverse transcriptions synthesize cDNA
UsingRT reagent kit (TaKaRa, article No. DRR037A) carry out cDNA reverse transcriptions, realTest operation to carry out by product description, concrete operations are as follows：
Using Reverse Transcriptase kit, converse record synthesis cDNA is carried out to 0.3 μ g total serum IgEs with RT Buffer.Using 10 μL reaction systems：5xPrimerScript Buffer 2μL、PrimeScript RT Enzyme Mix I 0.5μL、OligodTThe μ L of Primer 0.5, Random 6mers 0.5 μ L, RNA templates are 0.3 μ g and RNase Free dH2O is mended to 10 μ L.ObtainCDNA preserve that to put -20 DEG C of refrigerators standby.
2.3 Real-Time PCR
Using online primer-design software, CAPG gene orders are with reference to NCBI:NM_001256139.1, PTGIS gene sequenceRow are with reference to NCBI:Synthesized after NM_000961.3, interior participation in the election GAPDH, design of primers by invitrogen companies.Specific primer is such asShown in table 1：
The primer sequence table of table 1
WithPremix Ex TaqTMII (TaKaRa, article No. DRR081A) is expanded, and experimental implementation presses productSpecification is carried out.The μ L reaction systems of quantitative fluorescent PCR 20 are as follows：Premix Ex TaqTMII：10 μ L, primer(10μM)：Forward and reverse each 0.8 μ L, ROX Reference DyeII (50 ×) of primer：0.4 μ L, dH2O：6 μ L, template cDNA：2μL.Response procedures are carried out on ABI7500, and amplification program is：95 DEG C of 30sec, (95 DEG C of 5sec, 60 DEG C of 34sec) × 40 are followedRing.
3rd, statistical analysis
Real-time quantitative PCR amplification curve flex point understands that amplification curve entirety collimation is good, shows the amplification effect of each reaction tubeRate is close, and the limit is put down and without raising up now, exponent phase slope is larger, illustrates that amplification efficiency is higher；Sample amplified production is moltenSolution curve is all unimodal, illustrates that amplified production only has one, is specific amplification；According to qRT-PCR relative quantification formula：2-ΔΔCt× 100%, compare expression of the CAPG and PTGIS genes in scoliosis blood samples of patients and in control group blood.As a result show：The expression water of qRT-PCR stable amplification results, wherein CAPG and PTGIS genes in scoliosis blood samples of patientsFlat is respectively 43% and 40% in control group blood, as shown in Figure 1.Result above demonstrates high flux transcript profile expression dataConfluence analysis CAPG and PTGIS gene the result of downward is expressed in scoliosis patient.
The detection kit assembling of the scoliosis of embodiment 3
The kit of the present embodiment detection scoliosis includes consisting of part：
(1) blood sample, which extracts total serum IgE reagent, includes TRizol, chloroform, isopropanol, 75% ethanol and without enzyme water；
(2) Reverse Transcription includes：5x RT Buffers, SuperRT reverse transcriptases, dNTPS, OligodTPrimer, Random 6mers and RNase Free dH2O, the reverse transcription reaction liquid is included：250mM pH8.3 Tris-HCl, 375mM KCl, 15mM MgCl2, 50mM DTT.The consumption for carrying out 1 reverse transcription PCR is as shown in table 2, response proceduresFor：42 DEG C of 30min, 85 DEG C of 5min.
The Reverse Transcription system of table 2
| Component | Addition |
| 5x RT Buffers | 4μL |
| OligodT Primer(50μM) | 0.5μL |
| Random 6 mers(100μM) | 0.5μL |
| SuperRT reverse transcriptases (200U/ μ L) | 1μL |
| dNTPs(2.5mM) | 4μL |
| Total serum IgE | 1μg |
| RNase Free dH2O | To 20 μ L |
(3) quantitative PCR reagent includes：The primer sequence of table 1 in PCR Mix reaction systems, embodiment 2.The PCR MixThe component of reaction system includes reaction buffer, dNTPs, Mg2+, Ex TaqHS enzymes andGreen I.1 time is carried out to determineThe consumption for measuring PCR is as shown in table 3.Response procedures are 95 DEG C of 30sec, (95 DEG C of 5sec, 60 DEG C of 34sec) × 40 circulations.It is above-mentionedReagent can bought on the market.
The quantitative PCR system of table 3
| Component | Addition |
| PCR Mix reaction systems | 10μL |
| Sense primer (10 μM) | 0.5μL |
| Anti-sense primer (10 μM) | 0.5μL |
| Template cDNA | 2.0μL |
| Add sterile purified water | To 20 μ L |
Kit also includes：Positive control is that the blood rna or DNA and negative control of normal person are ddH2O。
One kit can include the consumption that above-mentioned each composition carries out multiple PCR, such as 25 times, 50 times, it is 100 inferior, respectivelyThe Specific amounts of composition depending on the circumstances or the needs of the situation depending on.
RNA extractions, reverse transcription are carried out to subject's sample into cDNA, according to optimal anti-using the reagent in mentioned reagent boxSystem enters performing PCR reaction, the control cDNA in quantitatively being detected as QPCR using the arm's length standard product in kit, inspection with conditionSurvey change of the expression quantity with respect to CAPG and PTGIS expression quantity in normal person of CAPG and PTGIS in subject's sample, analysis detectionAs a result, compare between sample and control and examined using t, P<0.05 is significant difference, is judged to detection sample positive.
The ELISA kit of the detection scoliosis of embodiment 4 is assembled and used
Conventional reagent in 1.ELISA experiments：
It is coated with buffer solution (pH9.6 carbonate buffer solution)：Na2CO31.59g, NaHCO32.93g, plus distilled water is extremely1L；
Lavation buffer solution (pH7.4)：8.0g NaCl；0.2g KH2PO4；2.9g Na2HPO4·12H2O；0.2g KCl；The Tween-20s of 0.5mL 0.05%, plus ddH2O to 1L；
Dilution:Bovine serum albumin(BSA) (BSA) 0.1g adds lavation buffer solution to 100mL；
The anti-human CAPG and PTGIS albumen mouse resource monoclonal antibody that the present invention is used is limited by Shanghai Fan Ke biotechnologiesCompany system is standby；
Protein standard substance CAPG and PTGIS are that people source recombinant protein is purchased from Shanghai Hu Zhen Industrial Co., Ltd.s.
2. it is prepared by polyclonal antibody：
The amino acid composition such as SEQ ID NO of CAPG and PTGIS antigens:7 and SEQ ID NO:Shown in 8, it can pass throughInduced expression is obtained, and can also be obtained by synthesis.
CAPG and PTGIS antigens are prepared by induced expression with reference to conventional method：Obtain coding CAPG and PTGIS DNAFragment, the fragment is inserted in pET plasmid vectors respectively, is then transformed into Escherichia coli and is built recombinant strains (pET-CAPG/BL21 and pET-PTGIS/BL21), recycle IPTG to carry out induced expression, separated by Ni-NTA affinity chromatographies pureChange fusion protein, obtain amino acid composition such as SEQ IDNO:CAPG antigens and amino acid composition such as SEQ IDNO shown in 7:8 institutesThe PTGIS antigens shown, antigen-immunized animal is can be used as after Purity is qualified；CAPG and PTGIS albumen after purification is moltenSolution takes appropriate protein solution to be mixed with Freund's complete adjuvant (Sigma) and emulsified, take 2Kg large ear rabbits, back skin in 1 × PBSLower multi-point injection antigen.After 2 weeks, take appropriate protein solution and incomplete Freund's adjuvant (Sigma) to mix and emulsify, subcutaneous,Second is carried out to be immunized.Later every 3 weeks, carry out third and fourth time and be immunized；Immune intravenous rabbit drop of blood degree is detected after being immunized through 4 times,Reach 1:After more than 50000, arteria carotis is carried out to immune rabbit and takes blood to collect immune whole blood, rabbit is collected after centrifugation blood is immunizedClearly；The rabbit immune serum for resisting CAPG and PTGIS albumen using Protein A affinity columns is purified, and prepares anti-CAPGWith the polyclonal antibody of PTGIS albumen, detect titre, specificity, sensitivity etc. of polyclonal antibody, obtain antiserum CAPG andPTGIS protein antibodies.
3. the coating of ELISA Plate：
The anti-coated ELISA Plate of CAPG and PTGIS rabbit polyclonal antibodies is prepared via a method which：With pH9.6 carbonHydrochlorate coating buffer solution will be diluted to the μ g/mL of purpose concentration 0.63 by anti-CAPG and PTGIS rabbit polyclonal antibodies after purification；Will dilutionGood antibody-solutions are added in micropore after mixing, 100 μ L/ holes, and 4 DEG C overnight；Board-washing 3 times, 200 μ L/ holes；Add 3%BSA closingsLiquid, 300 μ L/ holes, 4 DEG C are overnight；Board-washing 3 times, 200 μ L/ holes；- 20 DEG C of preservations.
4. the preparation of enzyme labelled antibody：
Anti- CAPG and PTGIS albumen mouse resource monoclonal antibody is taken, is coupled respectively with HRP, obtains enzymic-labelled antibody.TakeA certain amount of enzymic-labelled antibody is added in dilution, is fully mixed, is made its final concentration of 2 μ g/mL (can be according to specific conditionDepending on), 2-8 DEG C is kept in dark place.
5. the assembling of kit：
Detecting the ELISA kit of scoliosis includes the component in table 4：
The ELISA detection kit of table 4
Use for convenience, the kit can also include positive control：Normal human AB serum or the μ L of hyclone 500 and feminine genderControl：1%PBS 1mL.
The application method of the kit is as follows：
(1) CAPG and PTGIS standard proteins concentration respectively 900pg/mL, 600pg/mL, 300pg/mL are taken respectively,The ELISA Plate that the μ L of 150pg/mL, 75pg/mL solution 50 add coated antibody draws standard curve；
(2) blank well (blank control wells are not added with sample and enzyme marking reagent, and remaining each step operation is identical) is set respectively, treat test sampleSample wells, first adds the μ L of sample diluting liquid 40 on ELISA Plate in testing sample hole, the μ L of testing sample 10 are then added again, and (sample is finally diluteDegree of releasing is 5 times).Sample is added on ELISA Plate bottom hole portion by sample-adding, is not touched hole wall as far as possible, is gently rocked mixing；
(3) rearmounted 37 DEG C of shrouding film shrouding is used to incubate 30 minutes；
(4) carefully take shrouding film off, discard liquid, dry, cleaning solution is filled it up with per hole, discarded after standing 30 seconds, so weightIt is multiple 5 times, pat dry；
(5) the μ L of enzyme marking reagent 50 are added per hole；
(6) incubate, washing, step is ibid；
(7) the μ L of tmb substrate solution A 50 are first added per hole, add the μ L of tmb substrate solution B 50, gently concussion is mixed,37 DEG C of lucifuges develop the color 15 minutes；
(8) the μ L of terminate liquid 50, terminating reaction are added per hole；
(9) returned to zero, 450nm wavelength sequentially measures the absorbance (OD values) in each hole, calculated in each serum sample with blank wellProtein content.
The clinical detection of the ELISA kit of embodiment 5
1st, case
20 scoliosis patients for treating examination are chosen, are sampled during deriving from October, 2012 in December, 2015 in northThe medical patient of capital Concord Hospital orthopaedics.The blood sample of all research objects is gathered, rearmounted -80 DEG C of low temperature refrigerators is numbered and protectsDeposit.All clinical samples of this research, to patient know and inform and pass through through this Hospital Ethical Committee.
2nd, method
Sample disposal gathers venous blood 2mL, first room temperature 3000r/min with serum separation gel vacuum blood collection tube and centrifuges 10min,Upper serum is shifted, then at 4 DEG C, 16000 × g centrifugation 10min, the acellular serum packing in the upper strata often μ L of pipe 200 is drawn, puts -70DEG C preserve.Aforesaid operations are in completion in 2h after collection of specimens.Normal human AB serum uses implementation as positive control using in kitCAPG and PTGIS expressing quantities change with respect to normal human AB serum in ELISA kit detection blood sample in example 4.
3rd, result
As a result show, treat examination 20 preceding scoliosis patients blood sample in have in 7 clinical samples CAPG andExpression quantity is without significant difference in PTGIS expressing quantities and normal human AB serum；Have in 13 blood samples of patients samples CAPG andPTGIS expressing quantities are less than 60% of expression quantity in normal human AB serum, wherein have in 9 blood samples of patients samples CAPG andThe expression quantity of PTGIS albumen is less than the 40% of normal human AB serum's expression quantity.Through clinical further detection, 20 are treated examination9 preceding scoliosis are made a definite diagnosis in patient, this 9 preceding scoliosis patients are consistent with kit testing result prepared by the present invention.Infer accordingly, scoliosis patient before diagnostic kit of scoliosis can be distinguished clearly before this, and carried as clinicFor diagnostic clue.
Although above the present invention is described in detail with a general description of the specific embodiments,On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.CauseThis, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Beijing Sai Erweikang biomedicines Science and Technology Ltd.
<120>Application of the CAPG and PTGIS genes in scoliosis detection kit is prepared
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<160> 8
<170> PatentIn version 3.5
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Met Tyr Thr Ala Ile Pro Gln Ser Gly Ser Pro Phe Pro Gly Ser Val
1 5 10 15
Gln Asp Pro Gly Leu His Val Trp Arg Val Glu Lys Leu Lys Pro Val
20 25 30
Pro Val Ala Gln Glu Asn Gln Gly Val Phe Phe Ser Gly Asp Ser Tyr
35 40 45
Leu Val Leu His Asn Gly Pro Glu Glu Val Ser His Leu His Leu Trp
50 55 60
Ile Gly Gln Gln Ser Ser Arg Asp Glu Gln Gly Ala Cys Ala Val Leu
65 70 75 80
Ala Val His Leu Asn Thr Leu Leu Gly Glu Arg Pro Val Gln His Arg
85 90 95
Glu Val Gln Gly Asn Glu Ser Asp Leu Phe Met Ser Tyr Phe Pro Arg
100 105 110
Gly Leu Lys Tyr Gln Glu Gly Gly Val Glu Ser Ala Phe His Lys Thr
115 120 125
Ser Thr Gly Ala Pro Ala Ala Ile Lys Lys Leu Tyr Gln Val Lys Gly
130 135 140
Lys Lys Asn Ile Arg Ala Thr Glu Arg Ala Leu Asn Trp Asp Ser Phe
145 150 155 160
Asn Thr Gly Asp Cys Phe Ile Leu Asp Leu Gly Gln Asn Ile Phe Ala
165 170 175
Trp Cys Gly Gly Lys Ser Asn Ile Leu Glu Arg Asn Lys Ala Arg Asp
180 185 190
Leu Ala Leu Ala Ile Arg Asp Ser Glu Arg Gln Gly Lys Ala Gln Val
195 200 205
Glu Ile Val Thr Asp Gly Glu Glu Pro Ala Glu Met Ile Gln Val Leu
210 215 220
Gly Pro Lys Pro Ala Leu Lys Glu Gly Asn Pro Glu Glu Asp Leu Thr
225 230 235 240
Ala Asp Lys Ala Asn Ala Gln Ala Ala Ala Leu Tyr Lys Val Ser Asp
245 250 255
Ala Thr Gly Gln Met Asn Leu Thr Lys Val Ala Asp Ser Ser Pro Phe
260 265 270
Ala Leu Glu Leu Leu Ile Ser Asp Asp Cys Phe Val Leu Asp Asn Gly
275 280 285
Leu Cys Gly Lys Ile Tyr Ile Trp Lys Gly Arg Lys Ala Asn Glu Lys
290 295 300
Glu Arg Gln Ala Ala Leu Gln Val Ala Glu Gly Phe Ile Ser Arg Met
305 310 315 320
Gln Tyr Ala Pro Asn Thr Gln Val Glu Ile Leu Pro Gln Gly His Glu
325 330 335
Ser Pro Ile Phe Lys Gln Phe Phe Lys Asp Trp Lys
340 345
<210> 8
<211> 500
<212> PRT
<213>PTGIS antigens
<400> 8
Met Ala Trp Ala Ala Leu Leu Gly Leu Leu Ala Ala Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Ser Arg Arg Arg Thr Arg Arg Pro Gly Glu Pro Pro Leu
20 25 30
Asp Leu Gly Ser Ile Pro Trp Leu Gly Tyr Ala Leu Asp Phe Gly Lys
35 40 45
Asp Ala Ala Ser Phe Leu Thr Arg Met Lys Glu Lys His Gly Asp Ile
50 55 60
Phe Thr Ile Leu Val Gly Gly Arg Tyr Val Thr Val Leu Leu Asp Pro
65 70 75 80
His Ser Tyr Asp Ala Val Val Trp Glu Pro Arg Thr Arg Leu Asp Phe
85 90 95
His Ala Tyr Ala Ile Phe Leu Met Glu Arg Ile Phe Asp Val Gln Leu
100 105 110
Pro His Tyr Ser Pro Ser Asp Glu Lys Ala Arg Met Lys Leu Thr Leu
115 120 125
Leu His Arg Glu Leu Gln Ala Leu Thr Glu Ala Met Tyr Thr Asn Leu
130 135 140
His Ala Val Leu Leu Gly Asp Ala Thr Glu Ala Gly Ser Gly Trp His
145 150 155 160
Glu Met Gly Leu Leu Asp Phe Ser Tyr Ser Phe Leu Leu Arg Ala Gly
165 170 175
Tyr Leu Thr Leu Tyr Gly Ile Glu Ala Leu Pro Arg Thr His Glu Ser
180 185 190
Gln Ala Gln Asp Arg Val His Ser Ala Asp Val Phe His Thr Phe Arg
195 200 205
Gln Leu Asp Arg Leu Leu Pro Lys Leu Ala Arg Gly Ser Leu Ser Val
210 215 220
Gly Asp Lys Asp His Met Cys Ser Val Lys Ser Arg Leu Trp Lys Leu
225 230 235 240
Leu Ser Pro Ala Arg Leu Ala Arg Arg Ala His Arg Ser Lys Trp Leu
245 250 255
Glu Ser Tyr Leu Leu His Leu Glu Glu Met Gly Val Ser Glu Glu Met
260 265 270
Gln Ala Arg Ala Leu Val Leu Gln Leu Trp Ala Thr Gln Gly Asn Met
275 280 285
Gly Pro Ala Ala Phe Trp Leu Leu Leu Phe Leu Leu Lys Asn Pro Glu
290 295 300
Ala Leu Ala Ala Val Arg Gly Glu Leu Glu Ser Ile Leu Trp Gln Ala
305 310 315 320
Glu Gln Pro Val Ser Gln Thr Thr Thr Leu Pro Gln Lys Val Leu Asp
325 330 335
Ser Thr Pro Val Leu Asp Ser Val Leu Ser Glu Ser Leu Arg Leu Thr
340 345 350
Ala Ala Pro Phe Ile Thr Arg Glu Val Val Val Asp Leu Ala Met Pro
355 360 365
Met Ala Asp Gly Arg Glu Phe Asn Leu Arg Arg Gly Asp Arg Leu Leu
370 375 380
Leu Phe Pro Phe Leu Ser Pro Gln Arg Asp Pro Glu Ile Tyr Thr Asp
385 390 395 400
Pro Glu Val Phe Lys Tyr Asn Arg Phe Leu Asn Pro Asp Gly Ser Glu
405 410 415
Lys Lys Asp Phe Tyr Lys Asp Gly Lys Arg Leu Lys Asn Tyr Asn Met
420 425 430
Pro Trp Gly Ala Gly His Asn His Cys Leu Gly Arg Ser Tyr Ala Val
435 440 445
Asn Ser Ile Lys Gln Phe Val Phe Leu Val Leu Val His Leu Asp Leu
450 455 460
Glu Leu Ile Asn Ala Asp Val Glu Ile Pro Glu Phe Asp Leu Ser Arg
465 470 475 480
Tyr Gly Phe Gly Leu Met Gln Pro Glu His Asp Val Pro Val Arg Tyr
485 490 495
Arg Ile Arg Pro
500